projets

Indanio Fellowship

My objectives will be 1) to establish stable zebrafish lines expressing the human nuclear receptors not yet established by Indanio; 2) to de-orphanize at least three human nuclear receptors; 3) to lead initiatives in drug target identification and validation; and 4) to establish protocols for structural and pharmacokinetic analysis of potential drug targets. 1) Establishing the zebrafish lines will be accomplished using the Indanio-designed reporter system and standard molecular biology techniques. 2) The strategy to adopt orphaned nuclear receptors is a multistage project. My initial approach will be to prepare a single cell suspension from several thousand zebrafish embryos using the hydrolytic enzymes collagenase and dispase. The GFP-expressing cells containing ligand-bound nuclear receptors will be sorted from this suspension by flow cytometry (using the BD FACSAria). This enriched sample will then be lysed, and the His-tagged nuclear receptors purified by metal affinity chromatography. The purified protein, with bound ligand, will be subjected to two kinds of highly sensitive electrospray ionization mass spectrometry (using Q-Exactive ESI) to identify the ligand: Nondenaturing ESI to identify a total mass shift to the liganded protein, and more standard analysis of a CHCl3:MeOH lipid extraction. This protocol is novel and my own design.